银杏叶中异鼠李素对RAW264.7细胞向破骨细胞分化的影响及其分子机制

摘要

Objective To investigate the effect and potential molecular mechanisms of isorhamnetin (ISO) extracted from Ginkgo biloba on the differentiation of osteoclasts. Methods Osteoclast precursor RAW264.7 cells were induced with RANKL to differentiate into mature osteoclasts. Different concentrations of ISO were added to RAW 264.7 cells to determine its effect on osteoclast differentiation. CCK8 was used to evaluate the effect of ISO on cytotoxicity. The im-pact of ISO on the osteoclast differentiation process was investigated by analyzing tartrate resistance and bone resorption lacuna. Real-time PCR was performed to analyze the levels of differentiation marker genes, including tartrate resistant acid phosphatase (Trap), cathepsin K (Ctsk), and matrix metalloproteinase 9 (MMP-9);differentiation-related transcription factors, including the proto-oncogene protein c-Fos, nuclear factor of activated T-cells cytoplasmic 1(NFATc1);and the levels of downstream NF-κB p65 signaling pathway phosphorylation. Using the above-described method, we verified that ISO exerted an inhibitory effect on osteoclast differentiation and explored related molecular mechanisms. Results Dif-ferent concentrations of ISO (1-10 μM) had no cytotoxic effects on RAW264.7 cells, inhibited TRAP activity and decreased the number of bone resorption lacuna during osteoclast differentiation. When applied at a concentration of 10 μM, its inhibitory effect was significant. In addition, ISO significantly reduced the expression levels of Trap, Ctsk, MMP-9, c-Fos, NFATc1 and NF-κB p65 mRNA. Conclusion ISO extracted from Ginkgo biloba extract exerted an inhibitory effect on osteoclast differentiation, and the mechanism underlying its activity may involve the inhibition of the classical NF-κB pathway.%目的 探讨银杏叶中异鼠李素(isorhamnetin,ISO)对RAW264.7细胞向破骨细胞分化的影响及其可能的相关分子机制.方法 在核因子κB受体活化因子配基(receptor activator of NF-κB ligand,RANKL)诱导RAW264.7细胞分化为破骨细胞的同时,加入不同浓度的ISO(1～10μM),研究其对RAW264.7细胞向破骨细胞分化的影响,通过CCK8法检测及评估各作用浓度ISO的细胞毒性,通过抗酒石酸酸性磷酸酶(tartrate-resis-tant acid phosphatase staining,TRAP)染色及骨片吸收效果判定其对RAW264.7细胞向破骨细胞分化的影响.实时定量PCR检测破骨细胞分化过程的标志性基因:TRAP、组织蛋白酶K(cathepsin K,Ctsk)、金属基质蛋白酶9(matrix metallo protein,MMP-9);分化相关转录因子:原癌基因c-Fos(proto-oncogene protein,c-Fos)、核转录因子激活的T细胞1(nuclear factor of activated T-cells cytoplasmic 1,NFATc1)的表达及破骨细胞分化下游因子NF-κB p65信号通路的磷酸化水平.结果 不同浓度的ISO(1～10μM)对RAW264.7细胞无细胞毒性,并且能抑制TRAP活性,减少骨吸收陷窝数量,在浓度为10μM时最为显著;ISO能显著降低TRAP、Ctsk、MMP-9、c-Fos、NFATc1及NF-κB p65的mRNA表达.结论 ISO对RAW264.7细胞向破骨细胞的分化具有抑制作用,其机制可能与经典NF-κB通路的抑制有关.